The survival of cellular organisms is dependent on the physical properties of water. The freezing point of liquid water sets the lower limit for the survival of most cells, because the formation of ice causes dehydration and osmotic damage to the cell. Organisms that inhabit sub-zero environments, have special adaptations which permit the organism to survive. For example, Arctic and Antarctic fish which live in cold seawater have various macromolecular antifreeze polypeptides in the serum of their blood. Such antifreeze polypeptides are a mixture of glycoproteins having a range in relative molecular mass (Mr) from about 2,500 to 34,000 (antifreeze glycoproteins, or xe2x80x9cAFGPsxe2x80x9d) and antifreeze polypeptides (AFPs) with Mr from about 3,300 to 12,000. Ananthanarayanan (1989) Life Chemistry Reports 7:1-32 provides an overview of AFPs and AFGPs. See also DeVries (1983) Annu. Rev. Physiol 45: 245-260; Davies et al., (1990) FASEB J 4: 2460-2468 and Warren et al., U.S. Pat. No. 5,118,792.
At present, four distinct types of AFPs have been characterized from a variety of cold water fish See, Davies et al., (1990) FASEB J. 4: 2460-2468; and Griffith and Ewart et al. (1995) Bioteca Adv. 13(3): 375-402, and references therein. Type I AFPs are alanine-rich, xcex1-helical polypeptides, found in many right-eye flounders and sculpins. Type II AFPs are enriched with cysteine and are found in sea raven, smelt and herring. Type III AFPs are globular proteins found in several Zoarcoid families including eelpout and wolffish. Type IV AFPs are characterized by a helix bundle and have been found in longhorn sculpin, Myoxocephalus octodecimspinosis (see, G. Deng et. al. (1997) FEBS Letters 402: 17-20. Although the different AFPs and AFGPs are structurally distinct, they share the ability to inhibit ice crystal growth by binding to the ice surface.
AFPs in the liver (liver-type AFPs; Type I) from the Winter flounder, Pleuronectus americanus, have been studied extensively in terms of their structure and function, gene organization, gene expression and regulation. The genome of the Winter flounder contains multiple copies of liver or serum type AFP genes, most of which are arranged as regular tandem repeats (Scott et al., (1985) Proc. Natl. Acad Sci. USA. 82: 2613-2617).
WO97/28260 describes the presence of new isoforms of AFPs in the Winter flounder, Pleuronectes americanus that are synthesized in the peripheral tissues, such as the skin and gills. These AFPs are referred to as xe2x80x9cskin-type AFPsxe2x80x9d and are encoded by a distinct set of AFP genes that lack a signal peptide which is indicative of their intracellular location. Examples of skin isotypes from the Winter Flounder are wfsAFP-1 and wfsAFP-8. The presence of extracellular and intracellular AFPs with differential tissue expression within a single fish species has raised questions about the relative roles of these AFP isoforms in cold protection, function, and evolutionary relationship.
There exists a need in the art for new AFPs with unique physiologic function and in situ location that inhibit ice recrystallization and induce a concentration-dependent decrease in the freezing point of aqueous solutions. The present invention fulfills these and other needs.
The present invention relates to a new intracellular AFP found in the shorthorn sculpin (Myoxocephalus scorpius) (xe2x80x9csculpin-type AFPxe2x80x9d). These new sculpin-type AFPs aid the fish in its defense against the dangers of freezing in the ice-laden, sub-zero sea water. The shorthorn sculpin synthesizes AFPs that serve to depress the freezing temperature of its intracellular fluids. These sculpin-type AFPs thus function as protectors of intracellular components.
As such, the sculpin-type AFPs of the invention are generally useful in protecting solutions against freezing. This improves the shelf-life of many refrigerated foods, making the foods more palatable. The sculpin-type AFP, when added, inhibits ice recrystallization during cold storage, improving the texture and palatability of the food. In addition, cells that express the sculpin-type AFPs of the invention are more cold-tolerant than counterpart cells which do not express sculpin-type AFPs. Thus, the sculpin-type AFPs of the invention are used to improve the cold tolerance of bacteria, cell cultures, plants and animals. The sculpin-type AFPs of this invention can also be expressed in commercially farmed fish such as catfish, Atlantic salmon and talipia to improve the freeze tolerance of the fish. Sculpin-type AFPs also have certain antibacterial properties, providing a means of reducing unwanted bacteria in foods such as recombinant fruits expressing sculpin-type AFPs, and in blended food stuffs such as ice cream. This improves shelf life, food quality, and makes such products safer for consumption. These and other commercial applications are aspects of the present invention.
In one aspect, the present invention relates to an isolated intracellular antifreeze polypeptide (sculpin-type AFPs). In this embodiment, the polypeptide typically comprises four or more Pr-X2-Pr-X7 subsequences, where Pr is a polar amino acid and X is a naturally occurring or synthetic amino acid. The polypeptide is alanine rich, with X being predominately alanine. The sculpin-type AFPs of the present invention have the physical ability to induce a concentration-dependent decrease in the freezing point of an aqueous solution such as water.
The polypeptide typically comprises at least six of these 11 amino acid subsequences (note that the subsequences are overlapping) where the polar amino acids are N, D, E, and K. In addition, the preferred polypeptides have a MW of about 7900 DA to about 9700 DA. Typically, the sculpin-type AFPs are between about 45 and about 100 amino acids in length, more preferably between about 60 and 100 amino acids in length, and most preferably about 80-100 amino acids in length.
Preferred sculpin-type AFPs of the present invention are optionally assessed by examining the secondary structure of the polypeptides. In certain aspects, the polypeptides of the present invention, as measured by circular dichroism, are at least 70% xcex1-helical and, preferably at 0xc2x0 C., essentially entirely xcex1-helical. Certain polypeptides of the invention optionally do not meet these criteria, e.g., where the polypeptide is a fusion protein that includes subsequences that are unrelated to a sculpin-type AFP. Fusion proteins comprising sculpin-type AFP subsequences are a feature of the present invention.
Sculpin-type AFPs of the present invention are optionally defined by their immunological characteristics. Preferred sculpin-type AFPs bind polyclonal antibodies raised against the polypeptide shorthorn sculpin skin-type (sssAFP-2; SEQ ID NO:2). Preferred polypeptides also bind to polyclonal antibodies raised against the polypeptide sssAFP-2, where the polyclonal antisera are first subtracted with a skin-type polypeptide, such as wfsAFP-1, from the Winter Flounder.
In certain embodiments, polyclonal antisera for use in immunoassays are generated using sssAFP-2 as described herein. The polyclonal antisera is then tested for its cross-reactivity against skin-type AFPs from Winter flounder (e.g. wfsAFP-1 i.e., a skin isotype from the Winter flounder) using a competitive binding immunoassay. For example, the immunogenic polypeptide is immobilized to a solid support. wfsAFP-1 added to the assay competes with the binding of the antisera to the immobilized antigen. The ability of the skin-type AFPs from Winter flounder (wfsAFP-1) to compete for binding of the antisera to the immobilized protein is compared to the immunogenic polypeptide. The percent cross-reactivity for the above proteins is calculated, using standard calculations. Those antisera with less than 10% cross-reactivity with skin-type wfsAFP- 1 are selected and pooled. The cross-reacting antibodies are then removed from the pooled antisera by immuno absorbtion (subtraction) with wfsAFP-1. Preferred polypeptides are those that bind to the antisera raised against sssAFP-2 that has been subtracted with wfsAFP-1.
The isolated polypeptides are optionally present as purified lyophilized powders in aqueous solutions (e.g., comprising water, for instance with salts at physiological concentrations), in recombinant cells, plants, animals, bacteria, prokaryotes, cell extracts and the like. The polypeptides are optionally present in foods such as ice cream or frozen yogurt.
The isolated antifreeze polypeptides are optionally encoded by a coding nucleic acid (RNA or DNA) which hybridizes to a skin-type antifreeze nucleic acid encoding sssAFP-2 under high-stringency wash conditions. Preferably, the sculpin-type AFPs do not hybridize significantly to skin-type AFPs from Winter flounder under the same high-stringency wash conditions. In this aspect, the skin-type AFPs from Winter flounder do not significantly hybridize to a nucleic acid of the present invention, where the signal-to-noise ratio on a Southern or northern blot is reduced 75% or more, as compared to the binding of a fully complementary sculpin-type AFP. For example, if a radiolabeled nucleic acid probe from skin-type Winter flounder and a radiolabeled skin-type probe of this invention with the same specific activity are allowed to hybridize to duplicate Southern blots and an autoradiogram shows that the signal from the Winter flounder-specific probe has less than 25% the intensity of the skin-specific probe of this invention after a high-stringency wash, then the nucleic acid detected is a skin specific nucleic acid of this invention.
The present invention also provides nucleic acids such as expression vectors that encode sculpin-type AFPs. The sculpin-type AFPs encoded in the expression vector typically have the same properties as the isolated polypeptides described above and herein. Preferably, the expression vector encodes a skin-type intracellular antifreeze nucleic acid that hybridizes to a second skin-type, antifreeze sssAFP-2 nucleic acid in a Southern or northern blot under high-stringency conditions, but does not significantly hybridize to the nucleic acid wfsAFP- 1 from Winter flounder (skin isotype from the Winter flounder) under the same conditions. The skin-type intracellular antifreeze nucleic acid encodes a skin-type antifreeze polypeptide with the properties described above and herein. For example, the expression vector can encode a nucleic acid coding for polypeptide of SEQ ID NO:2.
The invention provides recombinant cells having a skin-type antifreeze nucleic acid that encodes a skin-type antifreeze polypeptide with the properties discussed above and herein. These cells typically express the sculpin-type AFP, but in certain embodiments, such as cells used in cloning, the cell does not necessarily express a sculpin-type AFP. Additionally, the nucleic acids are optionally linked to promoters active only under selected conditions. Cells expressing the polypeptide are cold resistant, meaning they can tolerate colder temperatures than similar cells which do not express a sculpin-type AFP, and can more readily survive freezing. The cell is optionally a eukaryotic cell such as a plant, fungal or animal cell, or is optionally a prokaryotic cell such as a bacterium, or is optionally an archaebacterial cell. It should be appreciated that cold tolerance is especially important in agriculture, aquaculture and food processing. Accordingly, cells for such applications are preferably transduced with sssAFP-2 nucleic acids as noted herein.
In still yet another aspect, the present invention relates to methods of depressing the freezing point of an aqueous composition by adding sculpin-type AFPs to the aqueous composition. Preferred sculpin-type AFPs of the invention depress the freezing point of an aqueous solution in a concentration-dependent manner. Additional methods of this invention relate to the inhibiting of ice recrystallization in certain compositions, especially foods such as diary products. The present invention also provides stabilization of certain biological and synthetic membranes using sculpin-type AFPs of this invention. In another embodiment, the present invention provides method for preserving cells, tissues and organs ex vivo using the sculpin-type AFPs, preferably, sssAFP-2.
In certain other aspects, the present invention provides antibodies that specifically bind to a skin-type antifreeze polypeptides herein. Preferred antibodies are specific for sssAFP-2. Indeed in one class of embodiments, the sculpin-type AFPs of the invention are optionally identified by binding to an antibody specific for a selected sculpin-type AFP. In addition to use in western blotting and immunoassay methods, a repeatable method of pooling antibodies is used for peptide identification.
In this aspect, an isolated intracellular antifreeze polypeptide where the polypeptide is selected based upon binding to a pool of subtracted polyclonal antibodies, where the original polyclonal antibodies are raised against the sssAFP-2 polypeptide from shorthorn sculpin and then subtracted with a different AFP such as wfsAFP- 1, from Winter flounder as previously described.
In other aspects, the present invention relates to an isolated nucleic acid encoding the isolated, intracellular antifreeze polypeptides described above and herein. Nucleic acids include s3-2 and s17-12.